Project Summary/Abstract Excessive recruitment of activated neutrophils to the lungs is one of the hallmarks of acute lung injury associated with sepsis. Sepsis is characterized by an intense systemic inflammatory response resulting in the release of early response cytokines, TNF and IL-1. These cytokines activate the endothelium leading to increased expression of adhesion molecules, chemokines and cytokines, critical mediators controlling neutrophil migration. Neutrophil migration from the circulation to inflamed tissue results in increased neutrophil survival and release of reactive oxygen intermediates. Understanding the molecular mechanisms regulating both neutrophil life span and pro-inflammatory mediator release during inflammation is critical for the development of therapies targeting neutrophil dysfunction. Studies from the current funding period demonstrated that cooperative signaling between TNF and [unreadable]2-integrins controls anti-apoptotic and pro- inflammatory signaling in adherent neutrophils. Our studies identified [unreadable]-PKC as an important regulator of cooperative signaling between these 2 receptors whereby [unreadable]-PKC acts as a signal transducer integrating information from the TNFR-1 receptor and [unreadable]2-integrins and this crosstalk leads to increased neutrophil survival and release of pro-inflammatory mediators. Thus, [unreadable]-PKC is a novel therapeutic target for the control of cytokine-mediated neutrophil dysregulation in inflammation. Whether this regulatory mechanism is unique to TNF or a common mechanistic pathway utilized by other proinflammatory mediators is not known and is the subject of this proposal. Inhibition of neutrophil apoptosis following migration through cytokine-activated endothelium is also [unreadable]2-integrin dependent suggesting common regulatory mechanisms. Using a physiologically relevant in vitro model system of neutrophil transmigration across human pulmonary microvascular endothelial cell (PMVEC) monolayers, we will determine whether [unreadable]-PKC is part of a common mechanistic pathway which controls cytokine-driven neutrophil adherence to endothelium, transmigration, and cellular activation. Mechanistic studies using a dominant negative [unreadable]-PKC TAT peptide in human neutrophils and [unreadable]-PKC siRNA in differentiated HL-60 (dHL-60) cells will ascertain the role of [unreadable]-PKC in regulating neutrophil transmigration and activity at inflammatory sites: We will: 1: Determine whether cytokine-mediated neutrophil transendothelial migration is [unreadable]-PKC-dependent and bacterial-mediated neutrophil transmigration is [unreadable]-PKC- independent, 2: Determine whether transendothelial migration suppresses neutrophil apoptosis through a [unreadable]- PKC-dependent mechanism. 3: Determine the mechanism by which [unreadable]-PKC regulates assembly of the NADPH oxidase and O2- generation. 4: Test the hypothesis that transendothelial migration regulates [unreadable]-PKC activity through phosphorylation which modifies [unreadable]-PKC localization and substrate specificity.